New sulfonamide-based glycosides incorporated 1,2,3-triazole as cytotoxic agents through VEGFR-2 and carbonic anhydrase inhibitory activity

New sulfonamide-triazole-glycoside hybrids derivatives were designed, synthesised, and investigated for anticancer efficacy. The target glycosides’ cytotoxic activity was studied with a panel of human cancer cell lines. Sulfonamide-based derivatives, 4, 7 and 9 exhibited promising activity against HepG-2 and MCF-7 (IC50 = 8.39–16.90 μM against HepG-2 and 19.57–21.15 μM against MCF-7) comparing with doxorubicin (IC50 = 13.76 ± 0.45, 17.44 ± 0.46 μM against HepG-2 and MCF-7, rescpectively). To detect the probable action mechanism, the inhibitory activity of these targets was studied against VEGFR-2, carbonic anhydrase isoforms hCA IX and hCA XII. Compoumds 7 and 9 gave favorable potency (IC50 = 1.33, 0.38 μM against VEGFR-2, 66, 40 nM against hCA IX and 7.6, 3.2 nM against hCA XII, respectively), comparing with sorafenib and SLC-0111 (IC50 = 0.43 μM, 53 and 4.8 nM, respectively). Moreover, the docking simulation was assessed to supply better rationalization and gain insight into the binding affinity between the promising derivatives and their targeted enzymes that was used for further modification in the anticancer field.

CA XII, CA XIV, and CA XV), some are cytosolic (CA I, CA II, CA III, CA VII, and CA XIII), mitochondrial (CA VA and CA VB), and secreted in milk and saliva (CA VI) 24,25 .
As a result of the hypoxic environment that cancer progression develops, extracellular hypoxic tumors become more acidic, which accelerates growth and metastasis of tumor 26 .Transmembrane hCA IX regulates extracellular and intracellular pH under hypoxic conditions, which is linked to the invasion and advancement of various malignancies, including brain, breast, lung, cervical, neck, colon and bladder cancers 27 .Also, hypoxic tumours typically exhibit increased resistance to conventional anticancer therapy as well as increased tumour aggression 28 .Consequently, hCA IX is presently viewed as a desirable anticancer target.Moreover, hCA XII is frequently overexpressed in aggressive tumours 29 .By limiting angiogenesis and metastasis, selective inhibition of hCA IX and XII activity has been shown to slow tumour growth and impact its ability to spread 30 .Therfore, targeting these isoenzymes in anticancer drug designs is essential due to their expression in hypoxic tumor cells 31 .
Sulfonamides are molecules with a wide range of biological activities that can potentially applied as possible therapeutic agents in drug development and discovery.Sulfonamides have wide applications as HIV protease inhibitors, antiviral 32 , antibacterial 33,34 , anti-inflammatory 35 , and anticancer drugs [36][37][38][39][40] .It is also widely known that certain sulfonamide derivatives have antimetabolite properties 41 .Additionally, it has been demonstrated that sulfonamide moieties combined with various heterocyclic ingredients impair the proliferation of human cancer cell lines especially breast cells 42,43 .Triazoles have emerged as key components in medicinal chemistry because of their many pharmacological applications like anticancer, antioxidant, antiviral, antitubercular, antibacterial, and anti-inflammatory activities 44 .Moreover, 1,2,3-triazoles have been discovered to be resistant to metabolic degradation and their presence in molecules may support an increase in their solubility and bioavailability, which advocate their utilization as pharmacophoric moieties 45 .
Potent VEGFR-2 inhibitor pazopanib I, which has sulfonamide moiety, has been developed and approved for the remediation of many types of cancer 16 .In 2021, Sayed et al. 46 found that sulfonamide derivative II combined with a 3,5-dioxopyrazolidine scaffold was highly cytotoxic through its inhibitory activity against VEGFR-2 (Fig. 1).SLC-0111, a ureido benzenesulfonamide molecule III, appears to be in Phase I/II clinical studies for advanced metastatic solid malignancies 47 and exerts its action through inhibition of carbonic anhydrase isoforms IX and XII.Incorporating benzenesulfonamide with 1,2,3-triazole scaffold in IV-VI exhibited considerable in vitro cytotoxicity against several human cancer cell lines by inhibiting VEGFR-2 and/or carbonic anhydrase activities [48][49][50] (Fig. 1).However, the addition of glycosides to heterocyclic compounds has generated significant hybrids with biologically valuable characteristics, particularly antiviral, anticancer, and antibacterial properties 51 .Compounds with triazole-glycoside motifs VII and VIII have demonstrated substantial cytotoxic and inhibitory impacts on EGFR, CDK-2, and/or VEGFR-2 52,53 (Fig. 1).
Considering the achievements of the past and our ongoing desire to identify efficacious chemotherapeutic targets [54][55][56][57] , a novel series of benzenesulfonamide-based targets bearing an azido group in 3, 4 or 1,2,3-triazoleglycoside scaffolds in 6-13 were were designed and synthesized through molecular hybridization of the three crucial cores: benzenesulfonamide, 1,2,3-triazole, and glycoside (Fig. 2).All synthesized compounds based on benzenesulfonamide were evaluated against cancer cell lines of the lung (A-549), liver (HepG-2), breast (MCF-7), and colon (HCT-116) To determine their action mechanism, the remarkable compounds were then assessed for their inhibitory potential against VEGFR-2 and the carbonic anhydrase isoforms hCA IX and hCA XII.They were additionally examined for their impact on the cell cycle, apoptosis, p-53, and the apoptotic proteins Bax and Bcl-2.Finally, the binding views within the chosen enzymes' active sites were predicted using in silico docking simulation.

Chemistry
The CuAAC reaction is known as copper-catalyzed cycloaddition of azides with terminal alkynes yields 1,4-disubstituted 1,2,3-triazoles and it was first reported in 2002 by Tornøe et al. 58 and Rostovtsev et al. 59 .In the present work, the synthesis of the designed target compounds benzene sulfonamides (6-13) containing triazole C-glycoside tails were achieved by Cu-catalyzed 1,3-DCR of the azido scaffold 3 and 4 with a panel of acylated O-propynyl glycosides 5a,b (Scheme1).
The synthetic route started with the synthesis of 4-azido-benzenesulfonamide derivatives 3 and 4 through the reaction of N-butylsulfanilamide 1 or N-cyclohexylsulfanilamide 2 60 with sodium azide.The IR spectrum of 4-azido derivatives 3 and 4 includes the disappearance of the absorption band for NH 2 and the appearance of new bands (2101 and 2100) cm -1 due to the formation of azide group respectively.
The synthesized azide derivatives 3 and 4 were reacted with the propargylated glycosides that; galacto, xylopyranosyl compound 5a,b, under click conditions by Cu-catalyzed cycloadition reaction (CuAAC) to achieve the 1,2,3-triazole glycosides derivatives 6-9 (Scheme 1).The required Cu(I) catalyst was generated by adding Naascorbate and copper sulfate which converted from Cu(II) in situ reaction at the medium to afford the targeted 1,2,3-triazole products.In the structures of the last glycosides, the sugar moiety was linked to C 4 of the triazole system, as analogs of modified C-nucleosides.The IR spectra of compounds 6-9 proved the characteristic bands related to (C=O) frequency in the acetate group.Their 1 H NMR spectra demonstrated the signals special to the sugar fragments which was appeared to the formed β-confirmation of the triazole-sugar linkage as proved by the coupling constant of the anomeric proton.The acetylated glycosides 6-9 derivatives was deprotected under the medium of a saturated ammonia solution in methanol and afforded the free hydroxyl triazole glycosides 10-13, which was approved with their spectral data.Disappearing of the acetyl-carbonyl absorption bands and appearing of the hydroxyl groups of the afforded deacetylated glycosidic compounds at their characteristic regions proved the structure.Finally, the NMR spectra signified the deactylation reaction and showed the existing of the hydroxyl protons signals and the disappearance of the signals of the methyl protons of the acetyl groups.

In vitro enzyme inhibitory assessment against VEGFR-2 and Carbonic anhydrase isoforms hCA IX and hCA XII
Aiming to elucidate action mechanism, the sulfonamide-based targets 4, 7 and 9 were selected for were selected for further evaluation of their in vitro inhibitory potency against VEGFR-2 and the carbonic anhydrase isoforms hCA IX and hCA XII due to their outstanding cytotoxic results.Their IC 50 values were recorded in Table 1 using sorafenib and SLC-0111 as references, respectively 64,65 .

Cell cycle arrest and apoptosis of compound 9
MCF-7 cells were treated for 24 h at a concentration of 21.15 μM to assess if the most potent cytotoxic derivative, benzenesulfonamide-1,2,3-triazole-glycoside 9, causes cell death using the apoptotic mechanism.The cells were further examined through flow cytometry with annexin-V staining (Figs. 5-7).Target 9 demonstrated higher cell accumulations of 29.98% during the G2/M phase than untreated MCF-7 cells, which indicated 10.39%, as shown in Fig. 6 and Table S6.The data obtained clearly illustrated that derivative 9 is able to arrest MCF-7 cells in the G2/M phase of the cell cycle.
The investigated benzenesulfonamide-1,2,3-triazole-glycoside 9 resulted in a significant increase to 15.77% in the early apoptosis from 0.35% (DMSO control) and a noticeable increase to 9.75% in the late apoptosis from 0.14% (DMSO control) with regard to apoptosis (Fig. 7 and Table S7).Furthermore, the derivative produced a 3.99% necrosis percentage as opposed to the DMSO control's 1.27%.Consequently, compound 9 may induce apoptosis, as suggested through a notable boost in apoptotic cells.

Impact of benzenesulfonamide-1,2,3-triazole-glycoside 9 upon Bax, Bcl-2 and p53 levels in MCF-7 cells
The two main mechanisms that control the cell during apoptosis are the extrinsic pathway, which is mediated by the death receptor, and the intrinsic pathway, which is mediated by the mitochondria 66 .The roles of Bcl-2 as an Scheme 1. Synthesis of 1,2,3-triazolyl hybrid glycosides.
anti-apoptotic and Bax as a pro-apoptotic (inducer) allow the two proteins to modify this programmed process, and the balance between them regulates cell death 67 .The tumor suppressor gene, p53, is an additional essential component that results in cell death or inhibits cell proliferation.Cancers that maintain their genomic stability and p53 inhibition may increased cell proliferation and become resistant to numerous anticancer treatments 68 .MCF-7 cells treated for 24 h with compound 9's IC 50 of 21.15 μM showed a 6.2-fold increase in Bax levels (271.45Pg/mL) compared to untreated control cells (43.66 Pg/mL).On the other hand, Bcl-2 protein was downregulated by 2.6 times in MCF-7 cells treated with compound 9, going from 8.51 to 3.27 ng/mL.Additionally, compound 9 boosted the p53 protein level by 7.4 times in MCF-7 treated cells compared to 125.40 Pg/mL in control cells (Table 3).

Molecular docking simulation
Docking simulation of benzenesulfonamide-1,2,3-triazole-glycosides 7 and 9 against VEGFR-2 and the carbonic anhydrase isoforms hCA IX and hCA XII was accomplished to establish a relationship through their activities and potential binding modes within the binding sites of the evaluated enzymes, based on the promising results obtained from the in vitro inhibitory assessment.
The docking processes were performed using the MOE-Dock (Molecular Operating Environment) software version 2014.0901 69,70.Initially, the procedures were verified through re-docking of the native ligands, sorafenib and acetazolamide, within the active sites of VEGFR-2, hCA IX and hCA XII (PDB codes: 4ASD, 3IAI and 1JG0, respectively) 64,71,72 .This resulted in energy score values of − 10.73, − 9.88 and − 9.65 kcal/mol with comparatively small values of RMSD (0.85, 1.22 and 1.16 Å, respectively), between the native ligands and their docked positions.www.nature.com/scientificreports/When compared to the original ligand, sorafenib, the screened 1,2,3-triazole-glycosides 7 and 9 displayed promising binding inside the VEGFR-2 active site, as shown in Fig. 8, with noted energy score values of − 9.45 and − 10.73 kcal/mol.The sulfonamide oxygen of both derivatives 7 and 9 afforded H-bond acceptors with the backbone of the key amino acid Cys919 (distance: 2.88 and 2.98 Å, respectively), resembling the parent ligand, sorafenib.Furthermore the acetyl oxygen of glycoside part in 7 formed H-bond acceptor with the backbone of Leu840 (distance: 3.04 Å).Removal of acetylmethyl fragment at p-6 of glycoside part in 9, pushed it away from Leu840 facilitated binding of triazole nitrogen with the backbone of Asn923 through H-bonding (distance: 2.74 Å).
Regarding to carbonic anhydrase isoforms hCA IX and hCA XII in Figs. 9 and 10, the sufonamide moiety in both 7 and 9 strengthened the binding within their active sites through generation of H-bond acceptor with the sidechain of Thr200 and ionic bond with Zinc ion.Additionally, the sidechain of Gln67 revealed H-bonding with acetyl oxygen in 9 within hCA IX and hCA XII, and with triazole nitrogen in 7 within hCA XII.Sulfonamide oxygen gave one H-bonding with His94 in 7 and two with Thr199 in 9 within hCA IX (distance: 3.22, 2.75 and 3.14 Å, respectively).
The incorporation of 1,2,3-triazole glycosides 7 and 9 with sulfonamide moiety provided significant fixing through several bonds inside the binding sites of VEGFR-2, hCA IX, and hCA XII.Through additional hydrophobic and hydrophilic interactions, the absence of the acetylmethyl fragment at position 6 of the glycoside portion in 9 encouraged its fitting with the active sites of the screening enzymes rather than its counterpart 7.   www.nature.com/scientificreports/

General experimental methods
Reichert Thermovar apparatus was used to recording the melting points which was uncorrected.Perkin-Elmer model 1720 FTIR spectrometer was used to recording the Infra-red spectra.Bruker AC-300 or DPX-300 spectrometer (500 MHz 1 H) (125 MHz 13 C) was used to investigate the Nuclear magnetic resonance.The values of δ ppm (chemical shifts) were registered compared to TMS as a standard reference.The coupling constants (J values) are offered in Hz.TLC holding aluminum silica gel 60 F245 was used to checking The advance of reaction completion.IR, 1 H NMR, 13 C NMR, Elemental analyses were measured at the National Research Center, Egypt.

Synthesis of azid 3 and 4 derivatives
Sulfanilamide derivatives 1 and 2 (0.007 mol) was dissolved in conc.acetic acid (18 mL) and distilled water (18 mL), then cooled in an ice bath until reach the temperature to (0-5) °C, then sodium nitrite solution (0.47 g, 0.007 mol in (5 mL) water) was added very slowly to the mixture and stirred for (10 min.).Finally, the solution of sodium azide (0.45 g, 0.007 mol in (25 mL) distilled water) was added drop-by-drop to the reaction mixture and stirred for (30 min.).The solid product was filtered off and washed with distilled water to obtain compounds 3 and 4. Copmpound 3 was previously synthesized 73 .

Table 2 .
In vitro Inhibitory assessment of the promising sulfonamide-based derivatives 4, 7 and 9 against VEGFR-2 and carbonic anhydrase isoforms hCA IX and hCA XII comparing with sorafenib and SLC-0111, respectively.IC 50 Compound concentration necessary to inhibit the enzyme activity by 50%, SD Standard deviation, each value is the mean of three values, (-) not detected.